17alpha-(2-carboxyethyl)-7, 17beta-dihydroxyandrost-4-en-3-one gamma-lactones and esters thereof



United States Patent 3,063,993 17a-(2 CARBOXYETHYL) 7,17/3 DIHYDROXYAN-DROST-4-EN-3-0NE 'y-LACTONES AND ESTERS THEREOF Robert C. Tweit,Wilmette, Ill., assignor to G. D. Searle & Co., Chicago, 111., acorporation of Delaware No Drawing. Filed Dec. 22, 1961, Ser. No.161,444 5 Claims. (Ci. 260-23957) This invention relates to17a-(2-earboxyethy1)-7,l76- dihydroxyandrost-4-en-3-one 'y-lactones andesters thereof, as also processes whereby these lactones and theiresters can be prepared. More particularly, this invention relates to newand useful chemical compounds having the formula wherein OR represents ahydroxy or alkanoyloxy substituent in the 7-position, either a or ,8, ofthe cyclopentanopolyhydrophenanthrene nucleus enformulated.

Among the alkanoyloxy radicals represented by OR, especially loweralkanoyloxy radicals are preferred, which is to say radicals having theformula the lower alkyl constituent being such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl,hexyl, isohexyl, heptyl, or like saturated, monovalent, acyclic,straightor branched-chain, hydrocarbon groupings having the formulawherein n represents a positive integer less than 8.

Equivalent to the foregoing lactones and their esters for the purposesof this invention are the corresponding hydroxy acids and their alkalisalts of the formula H3O (RHaCHzGOOM droxyandrost-4-en-3-one 'y-lactonewith either Gelasinospora telraspora or a species of Gliocladium,depending on whether a or ,8 configuration of the 7-hydroxy is desired;and the resultant alcohols, in turn, are converted to correspondingesters hereof by heating with pyridine and an appropriate alkanoic acidanhydride.

The fermentative procedure is carried out in a suitably nutritive mediumpreliminarily inoculated with the apposite culture and maintained atabout 25 with agitation under aerobic conditions conducive to thedevelopment of submerged growth. The starting material to behydroxylated is preferably added to the fluorishing culture insolution-for example, in acetone or propylene glycol-whereupon thefermentation is continued as before until such time as the hydroxylationis substantially complete-representatively, after upwards of 24 hours.The fermentation medium is then extracted with an appropriate solvent,such as dichloromethane, and the desired product obtained as the residuefollowing vacuum distillation.

Those skilled in the art will appreciate that the salts set forth abovereadily devolve from the corresponding lactones on contact with aqueousalkali, and the free acids are obtained from the salts by a criticallybrief exposure to a proton source. Prolongation of the exposure timeinduces lactonization.

The following examples describe in detail compounds illustrative of thepresent invention and methods whereby these compounds can be prepared.However, the invention is not to be construed as limited thereby, eitherin spirit or in scope, since it will be apparent to those skilled in theart of organic synthesis that many modifications, both of materials andof methods, may be practiced without departing from the purpose andintent of this disclosure. Throughout the examples hereinafter setforth, temperatures are given in degrees centigrade and relative amountsof materials in parts by Weight, except as otherwise noted. Specificrotations are at 25 and referred to the D line of sodium.

EXAMPLE 1 (2 carboxyethyl) 75,175 dihydroxyandrosta 4-en-3-0ne-lactone.A stainless steel fermentation tank is charged with a nutrientmedium containing, per 1000 parts of tap water, 33 parts of dextrose, 5parts of commercial cotton seed meal flour, 3 .parts of corn steepliquor, and 2 parts of silicone anti-foam emulsion. Tank and medium aresterilized by heating to temperatures in the range, ll0120, and thencooled to about 25 whereupon the medium is inoculated with an aqueoushomogenized culture of Gliocladium sp. A.T.C.C. 14513. The medium ismaintained at about 25 for 29 hours, during which time a stream ofsterile air is passed through, and the developing culture is agitated toproduce submerged growth. Sufficient17m-(Z-carbOXyethyD-UB-hydroxyandrost-4-en-3-one y-lactone (US.2,705,712) dissolved in a minimal quantity of acetone is then introducedto bring the concentration of lactone to 1 part per 3000 parts ofmedium. Agitation with aeration at about 25 is thereupon resumed for 12hours, at the end of which time the resultant mixture is extracted withdichloromethane. The extract is dried over anhydrous sodium sulfate andstripped of solvent by distillation. The residue is rinsed with hexaneand then taken up in benzene and chromatographed on silica gel, usingbenzene and ethyl acetate as developing solvents. From an eluatecomprising 40% ethyl acetate in benzene, upon evaporation of solvent, isobtained an oil which crystallizes when acetone is added thereto. Thiscrystalline material is l7ot-(2- carboxyethyl) 7,8,17/5dihydroxyandrost- 4 en 3 one 'y-lactone which, recovered on a filter anddried in air,

melts at 174-177 and has a specific rotation of +66". The product hasthe formula EXAMPLE 2 75 acetoxy 17a (2 carboxyethyl) 175hydroxyandrost-4-en-3-0ne 'y-lacton .A solution of 7 parts of 17a: (2earboxyethyl) 175,175 dihydroxyandrost 4- en-3-one 'y-lactone in amixture of 30 parts of pyridine and 20 parts of acetic anhydride isallowed to stand at room temperatures for 18 hours, then poured ontoice. A solid forms which is separated by filtration and recrystallizedfrom a mixture of acetone and ether to give 7 3 acetoxy 17a (2carboxyethyl) 17B hydroxyandrost-4-en-3-one 'y-lactone melting atapproximately 216-217 and with a specific rotation of +69. The

product has the formula Hal 0 OCOCH;

7 EXAMPLE 3 ized culture of Gelasinospom tetraspora A.T.C.C. 14512.

The medium is maintained at about 25 for 29 hours, during which time astream of sterile air is passed through and the developing culture isagitated to produce submerged growth. Suflicient 17a-(2-carboxyethyl)-17B-hydIoxyandrost-4-en-3-one y-lactone dissolved in a minimal quantityof acetone is then introduced to bring the concentration of lactone to 1part per 3000 parts of medium. Agitation with aeration at about 25 isthereupon resumed for 12 hours, at the end of which time the resultantmixture is extracted with dichloromethane. The extract is dried overanhydrous sodium sulfate and stripped of solvent by' distillation. Theresidue is washed by trituration with ether and then three timescrystallized from a mixture of ethyl acetate and ether to give17a-(2-carboxyethyl)-l7a,l7 B-dihydroxyandrost-4-en 3 one 7 lactonemelting at 26827-0 with decomposition and having a specific rotation of+43 .7. The product has the formula EXAMPLE 4 7oz acetoxy 17a (2carboxyethyl) 1713 hydroxyandrost-4-en-3-one 'y-lact0ne.-A mixture of 1part of 17a (2 carboxyethyl) 70:,175 dihydroxyandrost 4- en-3-one-lactone, 8 parts of pyridine, and 4 parts of acetic anhydride is letstand overnight at room temperatures and then poured into water. Theaqueous supernatant layer of the resultant mixture is decanted OE, andthe residue is dried and successively crystallized from aqueous methanoland a mixture of acetone and ether to give 700 acetoxy 17oz (2carboxyethyl) 175 hydroxyandrost-4-en-3-one 'y-lactone melting at152-156".

The product has the formula 0- OOOOH1 What is claimed is: l. A compoundof the formula lower alkanoyloxy radicals.

No references cited.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,063,993 November 13- 1962 Robert C. Tweit It is hereby certified thaterror appears in the above numbered patent requiring correction and thatthe said Letters Patent should read as corrected below.

Column 1, lines 18 to 28, column 3, lines 4 to 14 and lines 29 to 39 andcolumn 4 lines 4 to 14L lines 29 to 39, and lines 42 to 52, for thatportion of each formula reading column 3, line l8 for "l7[5 ,l7B-" read"(B ng line 65, for "-l'Zq Y l'IB- read Ja UB- Signed and sealed this31st day oi December 19630 (SEAL) Atte st: ERNEST W0 SWIDER I EDWIN L.REYNOLDS Attesting Officer Acting Commissioner of 7 Patents

1. A COMPOUND OF THE FORMULA